Opportunistic fungal pathogens, particularly Candida albicans, are a more serious problem as a result of the increasing number of immunocompromised individuals (cancer patients and AIDS victims). Two of the most commonly used antifungals are the polyenes (which interfere with membrane sterols) and azoles (which affect sterol biosynthesis). Although, the fungal ergosterol biosynthetic pathway has played an important role in our repertoire of antifungals, it is only poorly understood. The proposed research employs Saccharomyces and Candida to determine the sparking sterol requirements and bulk sterol requirements in these two yeasts, respectively. The sparking sterol function suggests that hormonal levels of sterol are involved in the transition from G1 and S in the cell cycle. The bulk membrane function requires that sterol meet the cell membrane requirements for membrane fluidity and permeability. In Saccharomyces cerevisiae, a number of Erg-non-auxotrophic mutations exist. However, there is disagreement as to the exact molecular configuration of the sparking sterol and the implication is that one or more of these erg mutations may be leaky. The cloning of three ERG genes has been undertaken. Molecular genetics involving gene disruption and allele replacement techniques which eliminate the ERG gene coding sequences should help resolve this controversy as to the structure of the sparking sterol. While Erg- non-auxotrophic mutations have been isolated for virtually each step in the Saccharomyces sterol pathway, only two different erg mutations have been conclusively isolated from Candida albicans. This proposal seeks to clone Candida ERG genes (EGR2,3,6) and by gene-disruption and allele replacement DNA technology ascertain whether bulk membrane requirements in this pathogen are more stringent than in Saccharomyces. A Candida gene library will be used to complement existing Saccharomyces erg mutations or the cloned Saccharomyces ERG genes will be used to isolate the cognate Candida genes by colony hybridization. Our long-range goals are to understand which sterol genes are essential, how they are regulated, and how sterols function with regard to the cell cycle. The elucidation of essential ergosterol biosynthetic steps which may be unique to Candida offers investigators an increased arsenal of strategies to combat fungal pathogens.